Serum alpha2-macroglobulin and cytokine measurements in an acute inflammation model in rats

An enzyme-linked immunosorbent assay (ELISA) for rat alpha2-macroglobulin (alpha2M) using a monoclonal antibody was developed, and used to measure alpha2M in sera from rats injected intramuscularly with turpentine oil as an inflammatory agent. The mean concentration of alpha2M gradually increased and peaked 2 days after the turpentine oil injection. The peak alpha2M concentration ranged from 2362-8472 microg/ml (mean 4531 microg/ml), which was 50-290 times higher than the pre-dosing levels of 23-61 microg/ml. In addition, interleukins (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon (IFN)-gamma were measured using commercial ELISA reagent kits. IL-6 and IL-8 increased and peaked 12 h after turpentine oil injection, the levels being 5-51 times and 2-38 times the pre-dosing ones, respectively. The concentrations of IL-1, IL-2, IL-4, IL-10 and IFN-gamma in rats injected with turpentine oil did not change.     

Maternal BMI Associations with Maternal and Cord Blood Vitamin D Levels in a North American Subset of Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study Participants

ObjectiveObesity in pregnancy may be associated with reduced placental transfer of 25-hydroxyvitamin D (25-OHD). The objective of this study was to examine associations between maternal BMI and maternal and cord blood levels of 25-OHD in full term neonates born to a single racial cohort residing at similar latitude. Secondary objectives were to examine associations between maternal glucose tolerance with maternal levels of 25-OHD and the relationship between cord blood 25-OHD levels and neonatal size.MethodsThis study was conducted among participants of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) Study meeting the following criteria: residing at latitudes 41–43°, maternal white race, and gestational age 39–41 weeks. Healthy pregnant women underwent measures of height, weight, and a 75-g fasting oral glucose tolerance test (OGTT) at approximately 28 weeks gestation. Maternal and cord blood sera were analyzed for total 25-OHD by HPLC tandem mass spectrometry. Statistical analyses included ANOVA and linear regression models.ResultsMaternal and cord blood (N = 360) mean levels (sd) of 25-OHD were 37.2 (11.2) and 23.4 (9.2) ng/ml, respectively, and these levels were significantly different among the 3 field centers (ANOVA p< 0.001). Maternal serum 25-OHD was lower by 0.40 ng/ml for BMI higher by 1 kg/m² (p<0.001) in an adjusted model. Maternal fasting plasma glucose, insulin sensitivity, and presence of GDM were not associated with maternal serum 25-OHD level when adjusted for maternal BMI. Cord blood 25-OHD was lower by 0.26 ng/ml for maternal BMI higher by 1 kg/m² (p<0.004). With adjustment for maternal age, field center, birth season and maternal serum 25-OHD, the association of cord blood 25-OHD with maternal BMI was attenuated. Neither birth weight nor neonatal adiposity was significantly associated with cord blood 25-OHD levels.ConclusionThese results suggest that maternal levels of 25-OHD are associated with maternal BMI. The results also suggest that interpretation of neonatal 25-OHD levels may need to incorporate specific maternal factors in addition to season of birth and latitude.     

Alpha 1-acid glycoprotein (AAG) levels in healthy and pregnant beagle dogs.

Serum alpha 1-acid glycoprotein (AAG) levels were measured in healthy beagles of various ages (66 male and 74 female) by turbidimetric immunoassay (TIA), and then separately--in pregnant beagles--by single radial immunodiffusion (SRID). The first experiment revealed that serum AAG levels ranged from 40 to 960 microg/ml (mean of 322 +/- 202 microg/ml) in male dogs, and from 47 to 833 microg/ml--in female dogs (mean of 316 +/- 199 microg/ml), without any significant sex- or age-related variation. The second experiment, however, revealed that serum AAG levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 microg/ml (mean of 634 +/- 246 microg/ml). In 7 of 8 dogs the AAG levels peaked about 45 days after ovulation. Despite a high value of 1,210-1,360 microg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40 microg/ml).     

Relationship between production of acute-phase proteins and strength of inflammatory stimulation in rats

The relationship between intensity of inflammatory stimulation and production of α(2)-macroglobulin (α2M) and α(1)-acid glycoprotein (AAG) in rats was investigated. Sprague-Dawley rats were injected with turpentine oil at doses of 0.05, 0.2 or 0.4 mL/rat. Serum levels of α2M, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured by enzyme-linked immunosorbent assay, and AAG was measured by single radial immunodiffusion. Peak serum levels of α2M and AAG in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. However, no significant differences were observed for peak serum levels of these acute-phase proteins between 0.2 and 0.4 mL/rat. Furthermore, peak serum levels of IL-6 and CINC-1 in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. Thus, the production of these acute-phase proteins has upper limits, even under increased strength of inflammatory stimulation in rats injected with turpentine oil.     

Expression of rat alpha 2-macroglobulin gene during pregnancy

Rat alpha 2-macroglobulin (alpha 2M) is a typical acute phase protein, the concentration of which in serum increases more than 100-fold after inflammation. It is also known that the protein increases during pregnant (and neonatal) stages. Using a specific cDNA probe, expression of the alpha 2M gene during pregnancy was studied at the mRNA level. During inflammation, the liver is almost the only organ producing alpha 2M, but during pregnancy the placenta and uterus were found to be major organs producing a large amount (70-80% of that of inflamed liver) of alpha 2M mRNA at days 12-15. The yolk sac, maternal liver and fetal (or neonatal) liver also produced a small but significant amount (5-20% of that of inflamed liver) of the mRNA. Southern blotting analysis showed that only one copy of the alpha 2M gene was present in a haploid rat genome. These results indicated that a single alpha 2M gene has the ability to respond to two completely-different physiological states.     

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.     

Cellular origin and induction of pregnancy-associated alpha 2-glycoprotein synthesis in pregnant rats

The synthesis of alpha 2-PAG was measured and compared in tissues and cells from normal non-pregnant females, and maternal and fetal rats in vitro to define the target cells hormonally regulated during pregnancy. Synthesis was measured by [L-14C]leucine incorporation into immunochemically isolated alpha 2-PAG and confirmed by radioimmunodiffusion. alpha 2-PAG synthesis was demonstrated in maternal peripheral blood leucocytes, placenta, breast, spleen, liver and fetal peripheral blood leucocytes and liver. Maternal and fetal liver were the most active tissue producers and fetal liver synthesized 4 times more alpha 2-PAG than did maternal liver. Furthermore, fetal peripheral blood leucocytes synthesized 2 times more alpha 2-PAG per cell than did these same maternal cells. A direct comparison of synthesis by cells from pregnant and non-pregnant female rats revealed that (1) maternal peripheral blood leucocytes synthesized 5 times more alpha 2-PAG per cell than did normal leucocytes, although these same cells synthesized approximately equal amounts of total cell protein per cell, (2) maternal peritoneal exudate macrophages also synthesized 5 times more alpha 2-PAG per cell than did macrophages obtained from normal female rats, and total protein synthesis by these cells also closely paralleled each other, (3) maternal and fetal plastic-adherent peripheral blood monocytes synthesized 22 and 58 times more alpha 2-PAG per cell respectively than did normal monocytes, (4) maternal and fetal non-adherent lymphocytes synthesized 8 and 16 times more alpha 2-PAG per cell respectively than did normal lymphocytes and (5) fetal monocytes and lymphocytes synthesized 3 and 2 times more alpha 2-PAG per cell than did maternal monocytes and lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical characteristics of alpha 2-globulin from the rat "pregnancy zone" in ontogeny

An antigen with electrophoretic mobility of alpha 2-globulins was found in the blood serum of the pregnant rats by means of electrophoresis. It is detected not only in the blood serum and tissue extracts of the pregnant rats, but occurs in low concentrations (1 microgram/ml) in the blood serum of the normal adult animals. The concentration of this protein attains the maximal values by the end of the pregnancy period. Alpha 2-globulin appears to be a protein associated with pregnancy.     

Induction of rat alpha 2-macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell-derived factor

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.     

Serum concentrations of calcitonin and parathormone in the cord blood of premature infants

Determinations of serum calcium (Ca), calcitonin (CT) and parathyroid hormone (PTH) were carried out in mixed cord blood of 23 preterm infants. Gestational age ranged between 25 and 37 weeks. 17 of theme were vaginally delivered while 6 were delivered by emergency Caesarean section. 4 neonates died because of respiratory distress syndrome. The serum was stored at -30 degrees C until the determinations. Serum Ca levels were determined by spectrophotometry while CT and PTH levels by RIA (Immuno Nuclear Co). In cord serum the mean (M +/- SE) Ca,CT and PTH concentrations of all neonates examined were respectively: 9,9 +/- 0,6 mg/dl; 176 +/- 44 pg/ml and 1100 +/- 446 pg/ml. Serum values of CT and PTH in preterm newborns delivered by emergency Caesarean section were significantly higher than in those neonates vaginally delivered (CT: 302 +/- 115 vs 94 +/- 9 pg/ml; p less than 0.005) (PTH:2655 +/- 1857 vs 466 +/- 59 pg/ml; p less than 0.05). No differences were observed between serum CT and PTH levels in preterm neonates of different gestational age. Both CT and PTH serum concentrations were higher in neonates who died. In conclusion, the preterm neonate is able to secrete both peptides and to maintain Ca homeostasis; the mode of delivery likely affects the CT and PTH secretion; unexplainable high CT and PTH serum levels were detected in poor outcome preterm infants.     

Effect of infusion of PGI-2, 6-keto-PGF-1 alpha and PGF-2 alpha on luteal function in the pregnant rat

Prostacyclin (PGI-2), 6-keto-PGF-1 alpha and PGF-2 alpha were infused continuously for 6 h into the dorsal aorta of rats 8 days pregnant. PGF-2 alpha (10 micrograms/h) significantly reduced plasma progesterone concentrations by 66% and luteal tissue concentrations of pregnenolone and progesterone by 78% and 95% respectively. Plasma concentrations of 20 alpha-dihydroprogesterone remained unchanged whilst luteal tissue concentrations rose 2-fold. Plasma progesterone concentrations were significantly reduced to 50% by PGI-2 (10 micrograms/h) but were unaffected by 6-keto-PGF-1 alpha (10 or 100 micrograms/h). Neither PGI-2 (10 micrograms/h) nor 6-keto PGF-1 alpha (10 or 100 micrograms/h) had any significant effect on plasma concentrations of 20 alpha-dihydroprogesterone or on luteal tissue concentrations of pregnenolone, progesterone or 20 alpha-dihydroprogesterone. Arterial blood pressure was unaffected by PGF-2 alpha and 6-keto-PGF-1 alpha, but was significantly reduced by PGI-2 at infusion rates greater than or equal to 60 micrograms/h.     

Comparison of bone metabolic markers between maternal and cord blood

We studied 41 normal pregnant women and their neonates in order to compare bone metabolism between them. We examined more specific bone formation markers (intact osteocalcin, bone-specific alkaline phosphatase) and a recently developed and more sensitive bone resorption marker (C-telopeptide of type I collagen; CTX) than previously available in maternal and umbilical cord venous blood taken at delivery. The concentrations of all markers of bone turnover, including CTX, in cord serum were significantly higher than those in maternal serum. There was no significant correlation between maternal and cord serum levels for any marker. These results indicate that fetal bone turnover is markedly enhanced compared with maternal bone turnover and is independent of maternal bone metabolism in late pregnancy.     

Renal reabsorptive capacity for alpha 2u-globulin in the adult male rat.

Adult male rats were maintained on 0, 5, 10, 15, and 20% casein diets to produce a series of animals having serum alpha 2u-globulin levels varying linearly from a normal of 31 micrograms/ml to a minimum of 13 micrograms/ml. In this way, it was possible to titrate endogenously the renal reabsorption and urinary excretion of this low molecular weight protein. The average maximal reabsorption rate (Tm) was established to be 9.7 micrograms/min and was reached at a renal filtered load (F alpha 2u) of 13.6 micrograms/min. These data were expressed in terms of a Tm:F alpha 2u ratio of 0.71. Below this value, the reabsorption declined from 70% to 50% of the F alpha 2u. Above 0.71, where F alpha 2u is less than the Tm, the reabsorption increased to 80-90%. It was observed that the fractional renal uptake of the alpha 2u-globulin varied linearly with the filtered load.     

Acute stimulatory effects of prostaglandin F2 alpha on serum progesterone concentrations in pregnant and pseudopregnant pigs.

The objective of this study was to investigate whether PGF2 alpha, administered to pregnant and pseudopregnant gilts in vivo, would cause an acute increase in serum progesterone concentrations prior to luteolysis. Pregnant (n = 9) and pseudopregnant (n = 4) gilts were fitted with a jugular vein cannula on day 40, were treated with 3 ml vehicle (control) i.m. on day 42 and with 15 mg PGF2 alpha on day 45. Blood samples were collected at frequent (5 and 15 min) intervals from 1 h before until 1 h after control and PGF2 alpha injections, at 15 min intervals for 4 h, and then at 5, 6, 9, 21, 33, 45 and 57 h post injection. Progesterone was measured by radioimmunoassay (RIA) in all samples. Porcine LH was measured by RIA in samples collected frequently in the 1 h pre- and 1 h post-injection periods. Serum progesterone concentrations were unchanged in both pregnant and pseudopregnant animals in response to control injection on day 42. However, in both pregnant and pseudopregnant gilts, PGF2 alpha injection on day 45 resulted in an acute increase (approximately 75-80% above pre-treatment levels; p less than 0.05) in serum progesterone lasting approximately 1 h, followed by a return to pre-treatment levels by 2 h, and then a decline to 1 ng/ml or less by 45-57 h (pregnant) or 21-57 h (pseudopregnant), associated with luteolysis. Serum LH concentrations were unchanged between 1 h pre- and post-treatment periods in response to either control or PGF2 alpha-treatment, in both pregnant and pseuodpregnant gilts. These results indicate that PGF2 alpha-injection produces a rapid and transient increase in serum progesterone concentrations which may result from a rapid and direct stimulatory action of PGF2 alpha on porcine luteal cell progesterone synthesis/secretion in vivo.     

Blood and placental concentrations of cadmium, lead, and mercury in mothers and their newborns

Placental transfer of cadmium, lead and mercury was studied under the conditions of environmental exposures of pregnant women to these heavy metals. Fifty pregnant women from industrial area and a similar control group from a semirural area were examined. Cadmium, lead and total mercury levels were determined in maternal erythrocytes and plasma, in placenta, and in erythrocytes and plasma of umbilical cord blood using atomic absorption spectrophotometry. Except for the cadmium plasma concentration in the control area, levels of the three metals were higher in maternal than in cord blood. The mean plasma values (arithmetic mean) of cadmium, lead, and mercury in industrial area were 0.53, 6.37, and 0.37 micrograms.100 ml-1 in maternal blood and 0.30, 4.82, and 0.31 micrograms.100 ml-1 in cord blood. Highest values of the correlation coefficients were found between the cadmium and mercury concentrations in maternal and cord blood erythrocytes. No striking effect of the place of residence of pregnant women on the heavy metal concentrations in biological materials could be found.     

Changes in serum lipids in rats treated with PGF2 alpha

Serum lipid concentrations were determined in rats treated with PGF2 alpha, PGE1, and controls. Administration of PGF2 alpha in rats influenced only the HDL lipid composition. HDL-cholesterol decreased while HDL-triglycerides increased. No significant difference was observed in the levels of serum total cholesterol, triglycerides, and phospholipids between animals treated with PGF2 alpha and controls. Reduced concentrations of serum lipid levels and especially of HDL-cholesterol, HDL-triglycerides, and HDL-phospholipids were found in the rats treated with PGE1. These results suggest that PGF2 alpha and PGE1 could modify serum lipid levels influencing lipoprotein metabolism.     

Rat alpha1-acid glycoprotein. Purification and immunological estimation of its serum concentration.

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.     

Sexual steroid levels and their clinical significance in the early neonatal age

Serum concentrations of progesterone (P) and estradiol (E2) in the umbilical cord blood were measured in healthy full term newborn infants and were related to the serum levels of indirect bilirubin at 72 hr postpartum in the same group of neonates. In those having higher concentration of bilirubin than 204 mumol/l at 72 hr after birth, cord P and E2 levels were significantly higher than in the rest of the group. Furthermore, strong correlation was found between serum levels of progesterone at 24 hr after birth and that of bilirubin at 72 hr postpartum in the same subjects. According to the authors' conclusion serum progesterone levels 24 hours after birth prognosticate the occurrence of neonatal hyperbilirubinaemia.     

Prostaglandin F2 alpha as the luteolysin in swine: VI. Hormonal regulation of the movement of exogenous PGF2 alpha from the uterine lumen into the vasculature

To test the endocrine-exocrine theory of maternal recognition of pregnancy in the pig 16 gilts were assigned randomly to a 2 X 2 factorial involving pretreatment with sesame oil (SO) or estradiol valerate (5 mg; EV) injected on Days 11 through 14 of the estrous cycle and an intrauterine injection of saline (5 ml; SA) or prostaglandin F2 alpha (50 micrograms; PGF) on Day 14. Peripheral blood samples were collected for 120 min postinjection and analyzed for 15-keto-13,14-dihydro-PGF2 alpha (PGFM). PGFM concentrations were lower in EV than SO gilts (438 vs. 844 pg/ml; p less than 0.05). There was heterogeneity of regression between EV and SO gilts (p less than 0.01), with EV gilts having a slower release of PGF from the uterine lumen into the vasculature. Prostaglandin F2 alpha did not increase mean PGFM concentrations (p greater than 0.10), but resulted in an altered temporal pattern of PGFM (p less than 0.05) compared to SA gilts. There was an interaction between the two treatments over time, with EV-PGF gilts demonstrating a slower, more gradual release of PGFM than SO-PGF gilts. To test whether prostaglandins of the E series were involved in this mechanism, gilts were assigned to two 4 X 4 latin squares balanced for residual effects and treated with saline or flunixen meglumine (Banamine). Each gilt was treated with four PGE:PGF infusion sequences (SEQ) in each uterine horn: phosphate-buffered saline (PBS; PBS-SEQ), PGE1 (50 micrograms), PGE2 (50 micrograms), and PGE1 (25 micrograms) + PGE2 (25 micrograms) (PGE-SEQ), with each infusion followed 15 min later by PGF (25 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)